Non-invasive Serological Monitoring for Crohn’s Disease Postoperative Recurrence

Abstract Introduction Crohn’s disease recurs after intestinal resection. This study evaluated accuracy of a new blood test, the Endoscopic Healing Index [EHI], in monitoring for disease recurrence. Methods Patients enrolled in the prospective POCER study [NCT00989560] underwent a postoperative colonoscopic assessment at 6 [2/3 of patients] and 18 months [all patients] following bowel resection, using the Rutgeerts score [recurrence ≥i2]. Serum was assessed at multiple time points for markers of endoscopic healing using the EHI, and paired with the Rutgeerts endoscopic score as the reference standard. Results A total of 131 patients provided 437 serum samples, which were paired with endoscopic assessments available in 94 patients [30 with recurrence] at 6 months and 107 patients [44 with recurrence] at 18 months. The median EHI at 6 months was significantly lower in patients in remission [Rutgeerts <i2] than those with recurrence; p = 0.033. The area under the receiver operating curve [AUROC] for EHI to detect recurrence at 6 months was comparable to that of faecal calprotectin [0.712 vs 0.779, p = 0.414]. EHI of <20 at 6 months had a negative predictive value of 75.7% (95% confidence interval [CI] 58.8–88.2), and sensitivity of 70% [95% CI 50.6–85.3] for detecting recurrence. Combining all time points, an EHI <20 had a negative predictive value of 70.3%. Changes in EHI significantly associated with changes in Rutgeerts scores over the 18 months. Conclusions The non-invasive multi-marker EHI has sufficient accuracy to be used to monitor for postoperative Crohn’s disease recurrence. A monitoring strategy that combines EHI with ileocolonoscopy, with or without faecal calprotectin, should now be prospectively tested.


Introduction
Within 1 year of an intestinal resection to remove all macroscopic Crohn's disease [CD], endoscopically identifiable disease recurrence occurs in up to 90% of patients, 1-3 and a further operation is required in up to 70% of patients within 10 years of their initial surgery. 4 The severity of endoscopic recurrence at the anastomosis 1 year after surgery is predictive of later clinical recurrence and the need for surgery. 5 Endoscopic assessment using ileocolonoscopy remains the gold standard for detection of recurrence, but is expensive, invasive, cannot be repeated frequently, and is not agreeable to many patients. 6 In the Post Operative Crohn's Endoscopic Recurrence [POCER] study, endoscopic monitoring and treatment of early disease recurrence reduced Crohn's disease progression. 7 Endoscopically identified disease recurrence and progression occur before clinical symptoms develop. 7 Measurement of serum C-reactive protein [CRP] is insensitive for disease recurrence, 8 partly because of the low disease burden with early recurrence and partly because some patients with active Crohn's disease do not develop an elevated C-reactive protein with active disease. Magnetic resonance enterography [MRE] is sensitive for diagnosing severe [Rutgeerts i3-i4] recurrence, but is less sensitive for the identification of early recurrent lesions. 8,9 Intestinal ultrasound has moderate sensitivity for detecting disease recurrence 10 but is dependent on visualisation of the anastomosis, body habitus of the patient, and the experience of the operator. Faecal calprotectin [FC] is currently the best studied biomarker of recurrence, and has good sensitivity and negative predictive value for detecting postoperative recurrence. [11][12][13][14] Many patients dislike stoolbased tests and would preferentially undergo a blood test instead of providing a faecal sample. 6,15 An accurate, simple, serum, non-invasive biomarker sensitive for disease recurrence would assist in the monitoring for disease recurrence after surgery and would improve patient compliance. Compliance with recommended postoperative endoscopic surveillance intervals is generally poor, with only 30-54% of patients undergoing a colonoscopy within 12 months of surgery. [16][17][18][19] The Endoscopic Healing Index [EHI; Monitr™ Panel] is a sensitive measure of intestinal mucosal damage and repair, validated against endoscopy, that combines 13 biomarkers of matrix remodelling, angiogenesis, cell adhesion, immune recruitment, and growth factors such as TGF-α. 20 The markers are combined with variable weightings to produce an endoscopic healing index [EHI] score that ranges from 0 to 100.
EHI has been previously validated in a prospective cohort of patients being treated for active Crohn's disease in the TAILORIX clinical trial, as well as a separate cohort from a tertiary clinical practice. 21,22 An index value of less than 20 was found to exclude the presence of endoscopic inflammation with a sensitivity of 83.2% to 97.1%. 20 A value of 50 or greater had a specificity of 87.8% to 100% for identifying the presence of endoscopic inflammation with a Simple Endoscopic Score for Crohn's Disease [SES-CD] ≥3. 20 Specificity of indices between 20 to 50 progressively increased as the values approached 50, indicating a higher probability of endoscopic inflammation with increasing EHI values.
The active disease setting, in which the goal is to detect mucosal healing in response to treatment, differs from the postoperative setting in which the goal is to detect the early recurrence of mucosal inflammation. This study assesses the utility of the Endoscopic Healing Index for the identification of early endoscopic recurrence in a well-characterised prospective cohort of Crohn's disease patients.

Clinical study
The POCER Study has been reported previously. 7 In this prospective study of 174 patients undergoing resection of all macroscopic Crohn's disease, 131 patients had at least one endoscopic assessment paired with a serum sample, and form the basis of this analysis. All patients underwent resection of all macroscopic luminal Crohn's disease [ Table 1] and had an endoscopically assessable primary anastomosis. 7 Patients were randomised [2:1 ratio] to a colonoscopy and endoscopic assessment at 6 months [active care] or to best standard drug therapy and no colonoscopy at 6 months. All patients underwent a colonoscopic assessment at 18 months postoperatively [Supplementary Figure 1].
Patients were classified according to the Montreal Classification. 23 Patients received drug therapy according to stratification based on pre-operative risk of recurrence and their study arm allocation. 7 Patients were deemed 'high risk' at POCER study entry if they had ≥1 of three risk factors: current smoking, penetrating disease phenotype, or previous surgical resection. Patients were deemed 'low risk' if they had none of these three risk factors for early disease recurrence. 7 For assessment of the endoscopic healing index, 437 serum samples were prospectively obtained: 118 at peri-operative baseline [prior to or within 6 weeks of surgery], 124 at 6 months, 88 at 12 months, and 107 at 18 months postoperatively; 131 patients (108 [82.4%] high risk) provided one or more serum samples and had at least one endoscopic assessment. A total of 77 patients provided all four serum samples. Baseline characteristics of the cohort are shown in Table 1.

Endoscopic assessments and changes in treatment
Endoscopic assessment was undertaken using the Rutgeerts score, 5 with recurrence defined as a score ≥i2, performed by  5 Patients in the active care arm with endoscopic recurrence at 6 months intensified medical therapy: low-risk patients commenced a thiopurine, and high-risk patients on a thiopurine from baseline commenced combination therapy with the addition of adalimumab 40 mg fortnightly. Patients on adalimumab monotherapy increased dosing to 40 mg weekly. 7   25 The logistic regression model developed [comprising the 13 best-performing markers] was then validated with data from the TAILORIX trial and a separate prospective cohort of CD patients. 26 Faecal calprotectin was measured using a quantitative enzyme immunoassay [fCAL™, Bühlmann, Schonenbuch, Switzerland], expressed as micrograms per gram of stool.

Endpoints
The primary analysis assessed the overall accuracy of the EHI score at various cut-offs for the presence or absence of endoscopic recurrence [Rutgeerts ≥i2] and for distinguishing between complete macroscopic mucosal normality [Rutgeerts i0] and severe recurrence [Rutgeerts i3 or i4].
Secondary outcomes of interest included other endoscopic scores, faecal calprotectin, and C-reactive protein [CRP]. The faecal calprotectin was defined as elevated or within the normal range based on our previous work in the postoperative setting, which established a sensitivity of 89% and specificity of 58% of calprotectin concentration 100 µg/g for recurrence of Rutgeerts i2 or greater. 12

Statistical analysis
Results are reported according to the Standards for Reporting of Diagnostic Accuracy Studies [STARD] statement. 27  The test comparison cohort included all samples that had simultaneous EHI, CRP, and calprotectin measurements at any time point.
The longitudinal cohort consisted of patients in the active care POCER study arm with EHI paired with a colonoscopy performed at both 6 and 18 months. The dynamics of EHI over time was assessed in patients who had a colonoscopy at both 6 and 18 months, to assess association with disease progression in those patients who did and those who did not increase their treatment intensity.

Demographics and baseline characteristics
Baseline patient demographics for the 131 patients are shown in Table 1. Of

Cross-sectional EHI in relation to Rutgeerts score
The median EHI tended to increase with increasing Rutgeerts score at both 6-and 18-month endoscopies [cross-sectional cohort; Figure 1].

Discussion
It is now accepted that proactive surveillance for postoperative recurrence is essential, and should commence within 6 months of resection. However, there are few non-invasive options with the sensitivity to identify minimal, localised, yet clinically significant postoperative disease.
This prospective longitudinal study has demonstrated that a multi-marker panel focused on gastrointestinal mucosal healing performs well in detecting or excluding recurrent disease.
Clinical indices in the postoperative setting, such as the Crohn's Disease Activity Index, are of little value as they do not correlate with the presence of recurrent endoscopic disease. 35 The use of blood-based biomarkers such as CRP to diagnose postoperative Crohn's disease has been disappointing as they lack sensitivity to diagnose localised disease, but patients prefer venepuncture over other tests. 6,15,36 Calprotectin has good sensitivity and specificity for disease activity but there are barriers to compliance, with up to 80% of patients preferring a blood test. 15 There are also a range of proposed thresholds for calprotectin in the postoperative setting. In our prospective large cohort, a threshold calprotectin concentration of 100 ug/g was optimal, with a sensitivity of 89%. A more recent meta-analysis reported an optimal threshold of 150 ug/g with a sensitivity of 81%. 12,37 Whereas ileocolonoscopy remains the gold standard for assessment of endoscopic disease activity, it cannot assess more proximal disease, is not always practical, cannot be performed frequently, and has the poorest acceptability to patients. 6 The EHI is still elevated immediately postoperatively. This most likely relates to the effect of surgery on this inflammatory index, even after the removal of diseased tissue. As a result, analysis of the change in the EHI between the postoperative and later analysis was not helpful. Its greatest value therefore lies in its use at 6 months postoperatively,  allowing discrimination between recurrence and maintenance of remission. The EHI was higher in patients with recurrence than those in remission at 6 months. EHI would therefore appear to be a good early marker of recurrence, with accuracy approaching that of faecal calprotectin. EHI did not discriminate as well at 18 months postoperatively. However, at 18 months the EHI differed significantly and discriminated between the clinically important macroscopic normality [i0], those with any macroscopic disease [≥i1], and those with severe endoscopic recurrence [≥i3]. The Rutgeerts scores of i2a five or more aphthous lesions or larger lesions confined to the ileocolonic anastomosis] or i2b [five or more aphthous lesions or larger lesions in the neoterminal ileum with normal intervening mucosa] were not assessed in this study, due to the low number of patients with i2a disease at 6 [five patients] and 18 months [four patients]. 38,39 Using the EHI threshold [<20] previously reported, at 6 months postoperatively the EHI had an acceptable sensitivity and negative predictive value which is concordant with previous data from general [non-surgical] Crohn's disease cohorts. 20 The upper threshold of EHI ≥50 had a high specificity, indicating that nearly all patients with this EHI value did indeed have recurrent endoscopic disease. Such a result would therefore indicate the need for colonoscopic confirmation. The overall AUROC at 6 months for the EHI was comparable to that of calprotectin, indicating the utility of EHI as an early marker of postoperative recurrence in Crohn's disease.
At 18 months, there was a lower sensitivity for the <20 threshold compared with 6 months, but the upper threshold of ≥50 showed a similar specificity to the 6-month value. The AUROC at 18 months for EHI was 0.52, with calprotectin being superior at this time point. The reasons for this difference in test performance between time points is unclear but may relate to specific markers within the EHI, such as those of matrix remodelling, being most relevant early in the postoperative period. In addition, the difference between EHI sensitivity at 6 and 18 months may relate to unknown confounders or to an increased rate of patient withdrawal between 6 and 18 months limiting sample size. There was also significant treatment heterogeneity in this cohort at later time points, mainly due to the active arm intensifying therapy after the 6-month endoscopic assessment.
At both time points, an EHI <10 ruled out any recurrent disease with a high negative predictive value [83. 3-100%] which is comparable to faecal calprotectin [100 µg/g, NPV of 90-93%] in this cohort. 12 Approximately 10% of patients in this cohort had a EHI of <10 across all time points. The very low false-negative rate means that such an EHI is reassuring that the patient does not have endoscopic recurrence.
We tested three thresholds for recurrence in this study, the standard Rutgeerts cut-off of <i2 vs ≥i2, i0 [mucosal normality] vs i3-4 [severe recurrence], and remission vs severe recurrence [i0-i1 vs i3-i4]. This tested the ability of the EHI to discern mild recurrence [lower disease burden] that may indicate progression, as well as the identification of already severe disease recurrence [higher disease burden]. Whereas recurrence can be ruled in at 6 months with an EHI ≥50, and an EHI <20 is reassuring for the absence of disease, patients who fall within this range should undergo further assessment for disease progression, with a faecal calprotectin or ileocolonoscopy. An EHI threshold of <10 is very sensitive for endoscopic recurrence and may allow avoidance of colonoscopy with a high level of confidence. These test thresholds therefore serve as clinically useful measures for excluding endoscopic disease recurrence, and serial EHI measurements may be helpful for prospectively monitoring changes in disease activity over time.
There is overall concordance between the EHI and the Rutgeerts score when all samples and time points are considered in a linear mixed model. Although the Rutgeerts score is unvalidated, it remains the best predictor of postoperative disease course. An additional advantage to being a minimally invasive test, the EHI may be elevated in the absence of disease seen on ileocolonoscopy, such as more proximal ileal disease. It therefore cannot be ruled out that a proportion of EHI false-positives reflect true proximal small bowel disease recurrence.
Limitations of this analysis include the smaller number of patients who had severe recurrent disease and colonic disease.
EHI can be used to monitor for endoscopic disease status in the postoperative setting despite low volume disease, with results similar to faecal calprotectin. The only other widely available serum marker, CRP, is not able to discern remission versus recurrence in any comparisons. The EHI demonstrates good utility to rule out postoperative recurrence, with a high sensitivity at a threshold of <20 and excellent test performance at a threshold of <10. Whereas use of the EHI is unlikely to replace postoperative endoscopic surveillance completely, it may enable a reduction in colonoscopy frequency in some patients. When used with ileocolonoscopy, with or without faecal calprotectin, a non-invasive multimodal approach could be used to monitor or disease recurrence. Such a model needs to be tested. For patients with the highest risk of recurrent disease, and therefore the greatest need for colonoscopy, it may have great value. In patients who decline ileocolonoscopy, the EHI test combined with faecal calprotectin measurement will provide early identification of patients with postoperative mucosal disease recurrence. This test adds to the diagnostic armamentarium after Crohn's disease surgery.

Conflicts of interest
T.D. and A.J. are paid employees of Prometheus Laboratories Inc. No other author has any conflict of interest. Prometheus had no input to the study design, results, or interpretation of data.

Author contributions
A.L.H., P.D.C., and M.A.K.: study concept and design; acquisition of data; analysis; data interpretation; drafting of the manuscript; critical revision of the manuscript for important intellectual content; statistical analysis; obtained funding. E.K.W.: acquisition of data; analysis; data interpretation; drafting of the manuscript; critical revision of the manuscript for important intellectual content. T.D. and A.J.: assay development, sample analysis, statistical analysis and critical revision of the manuscript for important intellectual content.

Data Availability
The de-identified data underlying this article will be shared on reasonable request to the corresponding author. Review by both the requester's and the authors' institutional Human Research Ethics Committee may be required.